PROJECT SUMMARY Prostate cancer is the second major cause of cancer death in American men. CBX7 is a Polycomb protein that participates in the assembly of the Polycomb repressive complex 1 (PRC1). CBX7 when associated with PRC1 has been shown to bind, through its chromodomain (ChD), to histone H3K27me3, a highly conserved epigenetic mark in all eukaryotes and trigger transcriptional repression of tumor suppressor p16INK4a. Elevated levels of CBX7 are directly linked to prostate cancer progression. Therefore, small molecules capable of modulating CBX7ChD-H3K27me3 interaction can serve a powerful research tool to study CBX7 functions in gene transcriptional regulation in biology and cancer. We have developed the ?first-in-class? small molecule inhibitors for the CBX7ChD. Our lead compound, MS351, is selective for CBX7ChD, and de-represses p16INK4a in prostate cancer cells through inhibiting CBX7 in its functionally active state. We recently developed novel MS351 analogs, of which some show much improved binding affinity over MS351. Accordingly, we have designed new Series I that combines the chemical features of our more potent analogs to develop potent and selective chemical probes for CBX7. Based on our recent metabolite identification study, we further designed Series II compounds to systematically block metabolic hotspots, while conserving existing binding, and probing for additional interactions in the CBX7ChD. Given that MS351 is selective for CBX7, we propose to develop the MS351-based, first-in-class PROTACs for CBX7 (Series III). Series I-III will be evaluated for target (CBX7ChD) engagement by the in vitro assays that our group has reported (FP and HSQC assay). In addition, for Series III, we will determine the cellular activity of these compounds in target protein CBX7 degradation in prostate cancer cell-lines (PC3). Global levels of CBX7 and other CBX proteins will be analyzed in these cells after compound treatment at different concentrations by using western blotting. This study will provide an estimate of potential selective degradation of the target protein CBX7, as well as determine whether CBX7 degradation by the PROTACS is dose-dependent. Overall, in this study, we will use structure-guided design to develop new chemical modulators (Series I-III) with improved potency, metabolic stability, and selectivity for CBX7ChD.